Dendritic cells are major initiators of immune responses, but are also responsible for the maintenance of peripheral T cell tolerance. A critical decision for induction of immune activation or immune tolerance is dependent on the activation status of DCs, which could be induced by the presence of diverse pathogens. For the recognition of pathogen associated molecular patterns (PAMPs) DCs express pattern recognition receptors (PRR), such as Toll-like receptors (TLR), or C-type lectin receptors (CLR). Previously, we have demonstrated, that steady-state DC subpopulations are specialized regarding processing and presentation of captured antigens. Therefore, DCs tightly control and direct activation of different T cell responses. Here, we investigated how TLR-mediated activation of murine cDC1 and cDC2 DCs influences the expression of molecules involved in the MHC class I antigen processing machinery. We provide evidence that activation of cDC2 DCs induces upregulation of Calreticulin, Calnexin, Erap1, ERp57, TAP1, TAP2, and Tapasin under certain immunostimulatory conditions in vivo. Importantly, delivery of targeted antigens to cDC2 DCs in combination with specific TLR-ligands allowed for cross-presentation and activation of CD8+ T cells in wildtype but also BATF3-/-(missing cDC1 DCs) mice in vivo. Moreover, the complete knock out of the three immunoproteasome subunits (β1i, β2i, β5i) hindered the cross-presentation of antigens by cDC2, but not cDC1 DCs. Our findings suggest that DCs harbor a strong flexibility in counteracting infections and tumor development if they are appropriately stimulated.

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8- DCs were critical for priming CD4+ T cell responses.

Dendritic cells (DCs) are central modulators of immune responses and, therefore, interesting target cells for the induction of antitumor immune responses. Ag delivery to select DC subpopulations via targeting Abs to DC inhibitory receptor 2 (DCIR2, clone 33D1) or to DEC205 was shown to direct Ags specifically to CD11c+CD8− or CD11c+CD8+ DCs, respectively, in vivo. In contrast to the increasing knowledge about the induction of immune responses by efficiently cross-presenting CD11c+CD8+ DCs, little is known about the functional role of Ag-presenting CD11c+CD8− DCs with regard to the initiation of protective immune responses. In this study, we demonstrate that Ag targeting to the CD11c+CD8− DC subpopulation in the presence of stimulating anti-CD40 Ab and TLR3 ligand polyinosinic-polycytidylic acid induces protective responses against rapidly growing tumor cells in naive animals under preventive and therapeutic treatment regimens in vivo. Of note, this immunization protocol induced a mixed Th1/Th2-driven immune response, irrespective of which DC subpopulation initially presented the Ag. Our results provide important information about the role of CD11c+CD8− DCs, which have been considered to be less efficient at cross-presenting Ags, in the induction of protective antitumor immune responses.

Background/Objectives The pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNF-α) directly activates the ribosomal S6 kinase RSK2 in vitro. We recently demonstrated the protective effect of RSK2 against TNF-induced bone loss. Interestingly, we found an increased activation of RSK2 in the joints of arthritis patients as well as in the inflamed joints of mice overexpressing the human TNF-α (hTNFtg). These observations prompted us to investigate the function of RSK2 in the development of TNF-α-induced inflammatory arthritis. Materials and Methods hTNFtg mice were crossed with RSK2-deficient (Rsk2y/-) mice. Clinical scoring and histomorphometry of the joints were assessed. We compared the levels of circulating pro-inflammatory cytokines as well as the cellularity of myeloid lineages in the spleen. The expression of cytokines and mesenchymal markers in the joints was determined via QPCR. Bone marrow transfer of Rsk2y/- and wild-type littermates into hTNFtg mice was performed and clinical scoring as well as histomorphometry of the joints was assessed. Primary fibroblast-like synoviocytes (FLS) from hTNFtg and hTNFtg; Rsk2y/ mice were isolated to analyse their expression of inflammatory cytokines and metalloproteinases as well as their proliferation and apoptosis in vitro. Results RSK2 deficiency in hTNFtg mice resulted in an early onset of clinical signs of arthritis as well as a drastic exacerbation of inflammation, increased cartilage destruction and increased local bone destruction. Increased levels of circulating pro-inflammatory cytokines and the increased proportion of all myeloid lineages in the spleen confirmed the enhanced inflammation in the hTNFtg mice lacking RSK2. Increased activation of synovial fibroblasts and macrophages in the joints of hTNFtg; Rsk2 y/- mice was demonstrated by the locally increased expression of pro-inflammatory cytokines and matrix metalloproteinases (MMPs). Importantly, the phenotype could not be transmitted by the transfer of Rsk2 y/- bone marrow into hTNFtg mice that demonstrated the essential role for RSK2 expression in mesenchymal cells driving the pathogenesis. In agreement, although no difference in the expression of pro-inflammatory cytokines or MMPs nor a change in apoptosis was detected in synovial fibroblasts isolated from hTNFtg; Rsk2 y/-, these cells displayed an increased proliferation rate. Conclusions The anti-proliferative function of RSK2 controls a cell autonomous negative feed-back against the activation of synovial fibroblasts by TNF-α, therefore limiting joint destruction in arthritis. Thus, activation of RSK2 is a potential target for the treatment of both local and systemic bone destruction in RA.

Background/Aims: Crucial steps in the initiation of lupus nephritis are the deposition of (auto-)antibodies and consequent complement activation. In spite of aggressive treatment patients may develop terminal renal failure. Therefore, new treatment strategies are needed. In extension to our previously published data we here analyzed the potential renoprotective mechanisms of bortezomib (BZ) in experimental lupus nephritis by focusing on morphological changes. Methods: Female NZB×NZW F1 mice develop lupus-like disease with extensive nephritis that finally leads to lethal renal failure. Treatment with 0.75 mg/kg BZ i.v. or placebo (PBS) twice per week started at 18 or 24 weeks of age. Antibody production was measured with ELISA and kidney damage was determined by quantitative morphological and immunohistochemical methods. Results: BZ treatment completely inhibited antibody production in both BZ-treated groups and prevented the development of nephritis in comparison to PBS-treated animals. Glomerular and tubulointerstitial damage scores, collagen IV expression, mean glomerular volume as well as tubulointerstitial proliferation and apoptosis were significantly lower after BZ treatment. Glomerular ultrastructure and in particular podocyte damage and loss were prevented by BZ treatment. Conclusions: BZ effectively prevents the development of nephritis in the NZB/W F1 mouse model. Specific protection of podocyte ultrastructure may critically contribute to renoprotection by BZ, which may also represent a potential new treatment option in human lupus nephritis.

Abstract AP-1 transcription factors are immediate early genes that sense changes in the cellular environment to control differentiation, proliferation and apoptosis. Their functions in B cells are poorly described and were only suggested by the pro-apoptotic role of JunB. Fra-1(FosL1), an interaction partner of JunB, has been firstly described in activated B cells. Here, we confirm that Fra-1 becomes up-regulated in activated murine primary B cells that prompted us to analyze B cell development and humoral immune responses following Fra-1 gain and loss of function in vivo. Mice over-expressing Fra-1 that displayed a reduced B2 B cell compartment failed to mount any IgG response and did not form germinal centers. Bone marrow transfer in mice lacking B cells demonstrated that these phenotypes were B cell autonomous. B cell-specific Fra-1 deficient mice developed enhanced secondary responses, further reinforcing a cell autonomous key role for Fra-1 in B cell function. In vitro experiments showed that sorted fra-1tg B2 cells that express all relevant genes required for plasma cell differentiation and class switching were unable to class-switch, do not proliferate and die after activation. Thus, we identified Fra-1 as negative regulator of IgG production and class switch that functions independently of chromatin remodeling and class switch gene expression. Our work therefore suggests that Fra-1 acts as a negative regulator of B2 B cell proliferation, which is required for Ig class switch.

OBJECTIVE:The proteasome inhibitor bortezomib has potent anti-myeloma and bone-protective activity. Recently, bortezomib was shown to directly inhibit osteoclastogenesis. The aim of this study was to analyze the influence and therapeutic effect of bortezomib in a mouse model of inflammatory arthritis.METHODS:Heterozygous human tumor necrosis factor α (hTNFα)-transgenic mice and their wild-type (WT) littermates were intravenously injected with 0.75 mg/kg of bortezomib or phosphate buffered saline twice weekly. The mice were assessed for clinical signs of arthritis. After 6 weeks of treatment, mice were analyzed for synovial inflammation, cartilage damage, bone erosions, and systemic bone changes. Osteoclast precursors from WT and hTNF-transgenic mice were isolated from bone marrow, treated with bortezomib, and analyzed for osteoclast differentiation, bone resorption, and expression of osteoclast-specific genes as well as apoptosis and ubiquitination.RESULTS:Bortezomib-treated hTNF-transgenic mice showed moderately increased inflammatory activity and dramatically enhanced bone erosions associated with a significant increase in the number of synovial osteoclasts. Interestingly, bortezomib did not alter systemic bone turnover in either hTNF-transgenic mice or WT mice. In vitro, treatment with therapeutically relevant concentrations of bortezomib resulted in increased differentiation of monocytes into osteoclasts and more resorption pits. Molecularly, bortezomib increased the expression of TNF receptor-associated factor 6, c-Fos, and nuclear factor of activated T cells c1 in osteoclast precursors.CONCLUSION:In TNF-mediated bone destruction, bortezomib treatment increased synovial osteoclastogenesis and bone destruction. Hence, proteasome inhibition may have a direct bone-resorptive effect via stimulation of osteoclastogenesis during chronic arthritis.

Heterozygous human tumour necrosis factor α transgenic (hTNFαtg) mice and wild-type littermates were intravenously injected with 0.75 mg/kg bortezomib or phosphate buffered saline twice a week. Mice were weekly assessed for clinical signs of arthritis. After 6 weeks of …

The proteasome inhibitor bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma. We recently demonstrated that bortezomib eliminates autoreactive plasma cells in systemic lupus erythematosus mouse models, thereby representing a promising novel treatment for Ab-mediated diseases. In this study, we investigated the effects of bortezomib on the just developing and pre-existing T-dependent Ab response toward dinitrophenyl-keyhole limpet hemocyanin and the T-independent type 2 response toward (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-Ficoll in BALB/c mice. Bortezomib treatment strongly reduced T-dependent Ab titers mainly due to depletion of plasma cells. In contrast, the early T-independent type 2 response against i.v. administered NIP-Ficoll, which is predominantly dependent on marginal zone (MZ) B cells, resisted bortezomib. Upon bortezomib treatment, immunoproteasome subunits and the antiapoptotic unfolded protein response including NF-κB were induced in NIP-Ficoll–stimulated MZ B cells, but not in plasma cells and follicular B cells. In summary, bortezomib treatment decreases Ab titers arising from T-dependent immune responses predominantly by eliminating plasma cells. In contrast, the early T-independent type 2 response protecting the organism against blood-borne pathogens remains largely intact due to a remarkable resistance of MZ B cells against proteasome inhibition.

Dendritic cells (DCs) are very important for the generation of long lasting immune responses against pathogens or the induction of anti-tumor responses. Targeting antigen to dendritic cells via monoclonal antibodies specific for DC cell surface receptors such as DEC205 was shown to elicit potent cellular and humoral immune responses in vivo. Therefore, we investigated whether this novel strategy might also be useful for the generation of new monoclonal antibodies against molecules of choice. We show, that by targeting the extracellular domain of the human C-type lectin receptor ClecSF6/DCIR/LLIR (hDCIR) to DEC205 on DCs in vivo, we were able to generate highly specific monoclonal antibodies against hDCIR.

Reinhard E Voll†, Kirsten Neubert, Silke Meister, Eva Guckel & Joachim R Kalden †Author for correspondence University of ErlangenNuremberg, Interdisciplinary Center for Clinical Research, IZKF-N2, Nikolaus-FiebigerCenter of Molecular Medicine, Gluckstrasse 6, 91054 Erlangen, Germany Tel.: +49 (0)9131 8539301 Fax: +49 (0)9131 8539311 rvoll@molmed.unierlangen.deand, University of ErlangenNuremberg, Department of Internal Medicine 3 (Rheumatology and Clinical Immunology), Krankenhausstrasse 12, 91054 Erlangen, Germany ‘Whenever pathogenic antibodies are predominantly secreted by long-lived plasma cells, good treatment responses become unlikely.’

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.

The proteasome inhibitor bortezomib, which induces cell death in various cancer cell lines including lymphatic neoplasias, has recently been approved for the treatment of relapsed multiple myeloma. Important mechanisms of proteasome inhibitor-mediated tumor cell death are the inhibition of NF- κ B activation and induction of the terminal unfolded protein response (UPR). However, little is known about effects of bortezomib on developing and mature lymphocytes. Therefore, Balb/C mice were injected with bortezomib and lymphocyte subsets were analyzed. This treatment resulted in dramatically decreased numbers of T and B lymphocyte precursors, while mature lymphocytes were only partially affected. Thymocytes were almost depleted 3 days after a single bortezomib injection, pro-B and pre-B cells already after 2 days. Thymocytes and B cell precursors recovered within 2 weeks. The decreased numbers of developing lymphocytes were due to apoptotic cell death accompanied by strongly increased caspase 3/7 activity. Within 8 h after bortezomib injection, there was a strong induction of heat shock protein 70 and C/EBP homologous protein in bone marrow B cells, indicating endoplasmic reticulum stress and activation of the terminal UPR, respectively. Hence, induction of apoptosis by proteasome inhibition can dramatically affect lymphocyte development, a fact which has important implications for the clinical use of bortezomib, especially in situations with ongoing lymphopoiesis.

Multiple myeloma is an incurable plasma cell neoplasia characterized by the production of large amounts of monoclonal immunoglobulins. The proteasome inhibitor bortezomib (PS-341, Velcade) induces apoptosis in various malignant cells and has been approved for treatment of refractory multiple myeloma. Inhibition of the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB) apparently contributes to the antitumor effects of bortezomib; however, this mechanism cannot fully explain the exceptional sensitivity of myeloma cells. Extensive protein synthesis as in myeloma cells is inherently accompanied by unfolded proteins, including defective ribosomal products (DRiPs), which need to be degraded by the ubiquitin-proteasome system. Therefore, we hypothesized that the proapoptotic effect of bortezomib in multiple myeloma is mainly due to the accumulation of unfolded proteins in cells with high protein biosynthesis. Using the IgG-secreting human myeloma cell line JK-6L and murine muH-chain-transfected Ag8.H myeloma cells, apoptosis induction upon proteasome inhibition was clearly correlated with the amount of immunoglobulin production. Preferentially in immunoglobulin-high myeloma cells, bortezomib triggered activation of caspases and induction of proapoptotic CHOP, a component of the terminal unfolded protein response induced by endoplasmic reticulum (ER) stress. In immunoglobulin-high cells, bortezomib increased the levels of proapoptotic Bax while reducing antiapoptotic Bcl-2. Finally, IgG-DRiPs were detected in proteasome inhibitor-treated cells. Hence, proteasome inhibitors induce apoptosis preferentially in cells with high synthesis rate of immunoglobulin associated with accumulation of unfolded proteins/DRiPs inducing ER stress. These findings further elucidate the antitumor activities of proteasome inhibitors and have important implications for optimizing clinical applications.